Influence of cold storage time on the protein changes and fatty acids deterioration of (Rutilus frisi kutum) during frozen storage

The main aim of this research was to identify fatty acids composition of Caspian sea of White fish Rutilus frisi kutum tissue and their changes during one year cold storage (-18Ċ).The secondary aim was to determine the changes of moisture, ash, protein, fat, and to investigate the effects of storage time on peroxide, TBAi, FFA, and extractability of myofibrillar proteins of the fish tissue during one year cold storage (-18 Ċ). 10 samples of (Rutilus frisi kutum) were randomly collected from Anzali landings. The samples were frozen at -30 Ċ and kept in cold storage at -18Ċ for one year. According to time table, the samples were examined. The results showed that 27 fatty acids were identified. The unsaturated fatty acids (UFA) and saturated fatty acids (SFA) were 74/09 and 21/63 %, respectively, in fresh tissue. So that DHA (C22:6) oleic acid (C18:1c) had high amounts (15/07 ,20/57 ) among the UFA and palmitic acid (C16:0) was the most (13/09 %) among the SFA. The effects of freezing and cold storage on fish tissue showed that UFA and SFA contents have reached to 58/79 and 22/17 %, respectively, at the end of cold storage. It indicated that these compound change to each other during frozen storage. Also ω-3 and ω-6 series of fatty acids was 24/22 and 15/56% in fresh tissue, but their contents decreased to 8/68 and 5/11% at the end of period. Among the fatty acids C22:6, C18:1c and C16:0 had the most changes. The changes of fatty acids were significantly at 95% level expected for C18:0. Results showed that moisture, ash, protein, and fat contents were 75/9±0/03, 1/28±0/012, 21/8±0/2, and 4/1±0/01 % respectively, in fresh tissue. The moisture, ash, protein, and fat contents were 72/3±0/04, 1/83±0/05, 1/91±0/01 and 19/9±0/01 % respectively, at the end of storage period. Lipid damage was measured on the basis of free fatty acids (FFA), peroxide value (PV), and Thiobarbituric acid index (TBA-i). PV, TBARS and FFA concentration of frozen Caspian Sea white fish stored at -18 Ċ the temporal variation of these three variables were statistically significant (p<0.001). Results of White fish myofibrillar proteins showed aggregation of bound reduced for stored at 12 months. SDS-PAGE analysis revealed that, the intensity of the myosin heavy chain and actin bound was reduced with increasing storage time. SDS-PAGE patterns showed that myosin heavy chain was much more susceptible to hydrolysis than actin. Key words: Rutilus frisi kutum, frozen storage, ω-3, ω-6, protein myofibrillar

Distribution of IgM, IgD and IgZ in mandarin fish, Siniperca chuatsi lymphoid tissues and their transcriptional changes after Flavobacterium columnare stimulation

The gene sequences of three different immunoglobulin (Ig) heavy chains, namely IgM, IgD and IgZ, were cloned from mandarin fish (Siniperca chuatsi) recently. In this study the distribution of these three kinds of Ig-producing cells in lymphoid-related tissues as head kidney, spleen, gill and intestine were investigated by using in situ hybridization, and their transcriptional changes were also analyzed by quantitative real-time PCR during 8 weeks after immunization. IgM-producing cells could be detected obviously and abundantly in all the tissues examined. A few numbers of IgD and IgZ positive cells were both detected in head kidney and spleen. IgZ positive cells could be detected in gill moderately while IgD showed negative results, otherwise no IgD or IgZ positive cells could be detected in intestine. After stimulated with bacterial pathogen Flavobacterium columnare G(4), the transcripts of these three Ig genes exhibited quite different kinetics. Significantly increased transcription of IgM gene was observed in almost all the tissues examined especially in boosted group. In contrast with IgM, seldom strong increase was examined for IgD and IgZ genes. For IgD, it seemed that the first injection could stimulate the immune response easier, since in almost all the tissues significant increase was detected at 1 or 2 weeks after injection. For IgZ, boosted injection could not enlarge the up-regulation of gene expression of first injection. This is the first case to report the transcriptional kinetics of three Ig genes in teleost after bacterin immunization. (C) 2008 Elsevier B.V. All rights reserved.

Preparation and mass spectrometric study of egg yolk antibody (IgY) against rabies virus

Rabies virus was used as the antigen to immunize laying chickens. Anti-rabies virus immunoglobulin Y(IgY) was isolated from yolks of the eggs laid by these chickens using a two-step salt precipitation and one-step gel filtration protocol. The purified IgY was reduced with dithiothreitol, and heavy chains (HC) and light chains (LC) were obtained. In addition, the purified IgY was digested with pepsin and the fragment with specific antigen binding properties (Fab) was produced. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOFMS), the average molecular weights of IgY, HC, LC, and Fab were determined as 167 250, 65 105, 18 660, and 45,359 Da, respectively. IgY has two structural differences compared with mammalian IgGs. First, the molecular weight of the heavy chain of IgY is larger than that of its mammalian counterpart, while the molecular weight of the light chain of IgY is smaller. Second, upon pepsin digestion, anti-rabies virus IgY is degraded into Feb, in contrast to mammalian IgG, which has been reported to be degraded into F(ab')(2) under the same conditions. Copyright (C) 2001 John Wiley & Sons, Ltd.

Expression of muscle-related genes and two MyoD genes during amphioxus notochord development

The notochord is one of the diagnostic features of the phylum Chordata. Despite the similarities in the early morphogenetic patterns of the notochords of various chordates, they are strikingly distinct from one another at the histological level. The amphioxus notochord is one example of an evolutionary novelty because it is made up of muscle cells. Our previous expressed sequence tag analysis, targeting messenger RNAs expressed in the adult amphioxus notochord, demonstrated that many muscle-related genes are expressed there. To characterize amphioxus notochord cells and to gain insights into the myogenic program in the notochord, we determined the spatial and temporal expression patterns of these muscle-related genes during amphioxus development. We found that BbNA1 (notochord actin), Amphi-Trop I (troponin I), Amphi-TPmyosin (tropomyosin), Amphi-MHC2 (myosin heavy chain), Amphi-nMRLC (notochord-specific myosin regulatory light chain), AmphinTitin/MLCK (notochord-specific titin/myosin light chain kinase), Amphi-MLP/CRP3 (muscle LIM protein), and Amphi-nCalponin (notochord-specific calponin) are expressed with characteristic patterns in notochord cells, including the central cells, dorsally located cells, and ventrally located cells, suggesting that each notochord cell has a unique molecular architecture that may reflect its function. In addition, we characterized two MyoD genes (Amphi-MyoD1 and Amphi-MyoD2) to gain insight into the genetic circuitry governing the formation of the notochord muscle. One of the MyoD genes (Amphi-MyoD2) is expressed in the central notochord cells, and the coexistence of Amphi-MyoD2 transcripts along with the Amphi-MLP/CRP3 transcripts implies the participation of Amphi-MyoD2 in the myogenic program in the notochord muscle.

Characterization and mapping of expressed sequence tags isolated from a subtracted cDNA library of Litopenaeus vannamei injected with white spot syndrome virus

A large number of polymorphic simple sequence repeats (SSRs) or microsatellites are needed to develop a genetic map for shrimp. However, developing an SSR map is very time-consuming, expensive, and most SSRs are not specifically linked to gene loci of immediate interest. We report here on our strategy to develop polymorphic markers using expressed sequence tags (ESTs) by designing primers flanking single or multiple SSRs with three or more repeats. A subtracted cDNA library was prepared using RNA from specific pathogen-free (SPF) Litopenaeus vannamei juveniles (similar to 1 g) collected before (0) and after (48 h) inoculation with the China isolate of white spot syndrome virus (WSSV). A total of 224 clones were sequenced, 194 of which were useful for homology comparisons against annotated genes in NCBI nonredundant (nr) and protein databases, providing 179 sequences encoded by nuclear DNA, 4 mitochondrial DNA, and 11 were similar to portions of WSSV genome. The nuclear sequences clustered in 43 groups, 11 of which were homologous to various ESTs of unknown function, 4 had no homology to any sequence, and 28 showed similarities to known genes of invertebrates and vertebrates, representatives of cellular metabolic processes such as calcium ion balance, cytoskeleton mRNAs, and protein synthesis. A few sequences were homologous to immune system-related (allergens) genes and two were similar to motifs of the sex-lethal gene of Drosophila. A large number of EST sequences were similar to domains of the EF-hand superfamily (Ca2+ binding motif and FRQ protein domain of myosin light chains). Single or multiple SSRs with three or more repeats were found in approximately 61 % of the 179 nuclear sequences. Primer sets were designed from 28 sequences representing 19 known or putative genes and tested for polymorphism (EST-SSR marker) in a small test panel containing 16 individuals. Ten (53%) of the 19 putative or unknown function genes were polymorphic, 4 monomorphic, and 3 either failed to satisfactorily amplify genomic DNA or the allele amplification conditions need to be further optimized. Five polymorphic ESTs were genotyped with the entire reference mapping family, two of them (actin, accession #CX535973 and shrimp allergen arginine kinase, accession #CX535999) did not amplify with all offspring of the IRMF panel suggesting presence of null alleles, and three of them amplified in most of the IRM F offspring and were used for linkage analysis. EF-hand motif of myosin light chain (accession #CX535935) was placed in ShrimpMap's linkage group 7, whereas ribosomal protein S5 (accession #CX535957) and troponin I (accession #CX535976) remained unassigned. Results indicate that (a) a large number of ESTs isolated from this cDNA library are similar to cytoskeleton mRNAs and may reflect a normal pathway of the cellular response after im infection with WSSV, and (b) primers flanking single or multiple SSRs with three or more repeats from shrimp ESTs could be an efficient approach to develop polymorphic markers useful for linkage mapping. Work is underway to map additional SSR-containing ESTs from this and other cDNA libraries as a plausible strategy to increase marker density in ShrimpMap.

Myocardial expression of a constitutively active alpha 1B-adrenergic receptor in transgenic mice induces cardiac hypertrophy.

Transgenic mice were generated by using the alpha-myosin heavy chain promoter coupled to the coding sequence of a constitutively active mutant alpha 1B-adrenergic receptor (AR). These transgenic animals demonstrated cardiac-specific expression of this alpha 1-AR with resultant activation of phospholipase C as shown by increased myocardial diacylglycerol content. A phenotype consistent with cardiac hypertrophy developed in adult transgenic mice with increased heart/body weight ratios, myocyte cross-sectional areas, and ventricular atrial natriuretic factor mRNA levels relative to nontransgenic controls. These transgenic animals may provide insight into the biochemical triggers that induce hypertrophy in cardiac disease and serve as a convenient experimental model for studies of this condition.

Idiotypic games within the immune network.

In this paper, we have considered the problem of selection of available repertoires. With Ab2 as immunogens, we have used the idiotypic cascade to explore potential repertoires. Our results suggest that potential idiotypic repertoires are more or less the same within a species or between different species. A given idiotype "à la Oudin" can become a recurrent one within the same outbred species or within different species. Similarly, an intrastrain crossreactive idiotype can be induced in other strains, even though there is a genetic disparity between these strains. The structural basis of this phenomenon has been explored. We next examined results showing the loss and gain of recurrent idiotypes without any intentional idiotypic manipulation. A recurrent idiotype can be lost in a syngeneic transfer and a private one can become recurrent by changing the genetic background. The change of available idiotypic repertoires at the B cell level has profound influences on the idiotypic repertoires of suppressor T cells. All these results imply that idiotypic games are played by the immune system itself, a strong suggestion that the immune system is a functional idiotypic network.

Loss of a major idiotype (CRIA) after repopulation of irradiated mice.

The normal immune response of A/J mice against arsonate coupled to hemocyanin is characterized by a major recurrent cross-reactive Id, the CRIA. This Id is encoded by a single gene segment combination: VHidcr11-DFL16.1e-JH2 for the H chain and Vkidcr-Jk1 for the L chain. In this report, we show that lethal irradiation of A/J mice followed by reconstitution with autologous or syngeneic lymphoid cells results in loss of major CRIA Id expression in the response to arsonate. Different protocols were performed to repopulate the irradiated mice. First, lethally irradiated A/J mice were reconstituted by the transfer of syngeneic bone marrow cells. Second, A/J mice were lethally irradiated while their hind limbs were partially shielded. Third, lethally irradiated A/J mice received a transfer of syngeneic spleen cells. The three groups of mice produce high titers of antiarsonate antibodies completely devoid of CRIA DH-JH related idiotopes expression. Moreover, a lack of affinity maturation is observed in the secondary antiarsonate response of all irradiated and reconstituted mice. A transfer of syngeneic peritoneal cells or a transfer of primed T cells in irradiated and reconstituted A/J mice do not restore in a significant manner either the recurrent CRIA expression or the affinity maturation of the antiarsonate response. Our data suggest that the choice of this Id is not solely dictated by the Igh locus.

Glucocorticoid induced muscle atrophy is associated with upregulation of myostatin gene expression

The mechanisms by which excessive glucocorticoids cause muscular atrophy remain unclear. We previously demonstrated that dexamethasone increases the expression of myostatin, a negative regulator of skeletal muscle mass, in vitro. In the present study, we tested the hypothesis that dexamethasone-induced muscle loss is associated with increased myostatin expression in vivo. Daily administration (60, 600, 1,200 micro g/kg body wt) of dexamethasone for 5 days resulted in rapid, dose-dependent loss of body weight (-4.0, -13.4, -17.2%, respectively, P <0.05 for each comparison), and muscle atrophy (6.3, 15.0, 16.6% below controls, respectively). These changes were associated with dose-dependent, marked induction of intramuscular myostatin mRNA (66.3, 450, 527.6% increase above controls, P <0.05 for each comparison) and protein expression (0.0, 260.5, 318.4% increase above controls, P <0.05). We found that the effect of dexamethasone on body weight and muscle loss and upregulation of intramuscular myostatin expression was time dependent. When dexamethasone treatment (600 micro g. kg-1. day-1) was extended from 5 to 10 days, the rate of body weight loss was markedly reduced to approximately 2% within this extended period. The concentrations of intramuscular myosin heavy chain type II in dexamethasone-treated rats were significantly lower (-43% after 5-day treatment, -14% after 10-day treatment) than their respective corresponding controls. The intramuscular myostatin concentration in rats treated with dexamethasone for 10 days returned to basal level. Concurrent treatment with RU-486 blocked dexamethasone-induced myostatin expression and significantly attenuated body loss and muscle atrophy. We propose that dexamethasone-induced muscle loss is mediated, at least in part, by the upregulation of myostatin expression through a glucocorticoid receptor-mediated pathway.

Endogenous expression and localization of myostatin and its relation to MHC distribution in C2C12 skeletal muscle cells

Myostatin is a negative regulator of skeletal muscle growth. We have previously reported that recombinant myostatin protein inhibits DNA and protein synthesis in C2C12 cells. Our objective was to assess if C2C12 cells express myostatin, determine its sub-cellular localization and the developmental stage of C2C12 cells in which myostatin mRNA and protein are expressed. To study the endogenous expression of myostatin, C2C12 myoblasts were allowed to progress to myotubes, and changes in the levels of endogenous myostatin mRNA expression were determined by RT-PCR. The myostatin protein and the two major myosin heavy chain (MHC) isoforms (MHC-I and -II) were determined by Western blot. Confirmation of the relative MHC expression patterns was obtained by a modified polyacrylamide gel electropheretic (PAGE) procedure. Imunofluorescence staining was employed to localize the site of myostatin expression and the relative distribution of the MHC isoforms. Co-expression of these proteins was studied using a dual staining approach. Expression of myostatin mRNA was found in myotubes but not in myoblasts. Myostatin protein was seen in most but not all, of the nuclei of polynucleated fibers expressing MHC-II, and myostatin was detected in the cytoplasm of myotube. The localization of myostatin protein in myotube nuclei was confirmed by Western blot of isolated nuclear and cytoplasmic fractions. Incubation of C2C12 myotubes with graded doses of dexamethasone dose-dependently increased the intensity of nuclear myostatin immunostaining and also resulted in the appearance of cytoplasmic expression. In conclusion, myostatin was expressed mostly in C2C12 myotubes nuclei expressing MHC-II. Its predominant

Neuropeptide Y Y(1) receptor regulates protein turnover and constitutive gene expression in hypertrophying cardiomyocytes.

Increased levels of neuropeptide Y correlate with severity of left ventricular hypertrophy in vivo. At cardiomyocyte level, hypertrophy is characterised by increased mass and altered phenotype. The aims were to determine the contributions of increased synthesis and reduced degradation of protein to neuropeptide Y-mediated increase in mass, assess effects on gene expression, and characterise neuropeptide Y Y receptor subtype involvement. Neuropeptide Y (10 nM) increased protein mass of adult rat ventricular cardiomyocytes maintained in culture (24 h) (16%>basal) and de novo protein synthesis (incorporation of [14C]phenylalanine) (18%>basal). Neuropeptide Y (100 nM) prevented degradation of existing protein at 8 h. Actinomycin D (5 µM) attenuated increases in protein mass to neuropeptide Y (=1 nM) but not to neuropeptide Y (10 nM). [Leu31, Pro34]neuropeptide Y (10 nM), an agonist at neuropeptide Y Y1 receptors, increased protein mass (25%>basal) but did not stimulate protein synthesis. Neuropeptide Y-(3–36) (10 nM), an agonist at neuropeptide Y Y2 receptors, increased protein mass (29%>basal) and increased protein synthesis (13%>basal), respectively. Actinomycin D (5 µM) abolished the increase in protein mass elicited by neuropeptide Y-(3–36) but not that by [Leu31, Pro34]neuropeptide Y. BIBP3226 [(R)-N2-(diphenylacetyl)-N-(4-hydroxyphenylmethyl)-d-arginine amide] (1 µM), a neuropeptide Y Y1 receptor subtype-selective antagonist, and T4 [neuropeptide Y-(33–36)]4, a neuropeptide Y Y2 receptor subtype-selective antagonist, attenuated the increase in protein mass to 100 nM neuropeptide Y by 68% and 59%, respectively. Neuropeptide Y increased expression of the constitutive gene, myosin light chain-2 (MLC-2), maximally at 12 h (4.7-fold>basal) but did not induce (t=36 h) expression of foetal genes (atrial natriuretic peptide (ANP), skeletal-a-actin and myosin heavy chain-ß). This increase was attenuated by 86% and 51%, respectively, by BIBP3226 (1 µM) and T4 [neuropeptide Y-(33–36)]4 (100 nM). [Leu31, Pro34]neuropeptide Y (100 nM) (2.4-fold>basal) and peptide YY-(3–36) (100 nM) (2.3 fold>basal) increased expression of MLC-2 mRNA at 12 h. In conclusion, initiation of cardiomyocyte hypertrophy by neuropeptide Y requires activation of both neuropeptide Y Y1 and neuropeptide Y Y2 receptors and is associated with enhanced synthesis and attenuated degradation of protein together with increased expression of constitutive genes but not reinduction of foetal genes.

Proteomics on porcine haptoglobin and IgG/IgA show protein species distribution and glycosylation pattern to remain similar in PCV2-SD infection

Haptoglobin (Hp) and immunoglobulins are plasma glycoproteins involved in the immune reaction of the organism after infection and/or inflammation. Porcine circovirus type 2-systemic disease (PCV2-SD), formerly known as postweaning multisystemic wasting syndrome (PMWS), is a globally spread pig disease of great economic impact. PCV2-SD affects the immunological system of pigs causing immunosuppression. The aim of this work was to characterize the Hp protein species of healthy and PCV2-SD affected pigs, as well as the protein backbone and the glycan chain composition of porcine Hp. PCV2-SD affected pigs had an increased overall Hp level, but it did not affect the ratio between Hp species. Glycoproteomic analysis of the Hp β subunits confirmed that porcine Hp is N-glycosylated and, unexpectedly, O-glycosylated, a PTM that is not found on Hp from healthy humans. The glyco-profile of porcine IgG and IgA heavy chains was also characterized; decreased levels of both proteins were found in the investigated group of PCV2-SD affected pigs. Obtained results indicate that no significant changes in the N- and O-glycosylation patterns of these major porcine plasma glycoproteins were detectable between healthy and PCV2-SD affected animals.

PKC-β exacerbates in vitro brain barrier damage in hyperglycemic settings via regulation of RhoA/Rho-kinase/MLC2 pathway

Stroke patients with hyperglycemia (HG) develop higher volumes of brain edema emerging from disruption of blood-brain barrier (BBB). This study explored whether inductions of protein kinase C-β (PKC-β) and RhoA/Rho-kinase/myosin-regulatory light chain-2 (MLC2) pathway may account for HG-induced barrier damage using an in vitro model of human BBB comprising human brain microvascular endothelial cells (HBMEC) and astrocytes. Hyperglycemia (25 mmol/L D-glucose) markedly increased RhoA/Rho-kinase protein expressions (in-cell westerns), MLC2 phosphorylation (immunoblotting), and PKC-β (PepTag assay) and RhoA (Rhotekin-binding assay) activities in HBMEC while concurrently reducing the expression of tight junction protein occludin. Hyperglycemia-evoked in vitro barrier dysfunction, confirmed by decreases in transendothelial electrical resistance and concomitant increases in paracellular flux of Evan's blue-labeled albumin, was accompanied by malformations of actin cytoskeleton and tight junctions. Suppression of RhoA and Rho-kinase activities by anti-RhoA immunoglobulin G (IgG) electroporation and Y-27632, respectively prevented morphologic changes and restored plasma membrane localization of occludin. Normalization of glucose levels and silencing PKC-β activity neutralized the effects of HG on occludin and RhoA/Rho-kinase/MLC2 expression, localization, and activity and consequently improved in vitro barrier integrity and function. These results suggest that HG-induced exacerbation of the BBB breakdown after an ischemic stroke is mediated in large part by activation of PKC-β.

CD38 expression level and pattern of expression remains a reliable and robust marker of progressive disease in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) follows a variable clinical course which is difficult to predict at diagnosis. We assessed somatic mutation (SHM) status, CD38 and ZAP-70 expression in 87 patients (49 male, 38 female) with stage A CLL and known cytogenetic profile to compare their role in predicting disease progression, which was assessed by the treatment free interval (TFI) from diagnosis. Sixty (69%) patients were SHM+, 24 (28%) were CD38+ and ten (12%) were ZAP-70+. The median TFI for: (i) SHM + versus SHM- patients was 124 versus 26 months; hazard ratio (HR) = 3.6 [95% confidence interval (CI) = 1.8 - 7.3; P = 0.001]: (ii) CD38- versus CD38+ patients was 120 versus 34 months; HR = 2.4 (95% CI = 1.4 - 5.3; P = 0.02); and (iii) ZAP70- versus ZAP70+ was 120 versus 16 months; HR = 3.4 (95% CI = 1.4 - 8.7; P = 0.01). SHM status and CD38 retained prognostic significance on multivariate analysis whereas ZAP-70 did not. We conclude that ZAP-70 analysis does not provide additional prognostic information in this group of patients.

Clathrin is spindle-associated but not essential for mitosis

BACKGROUND: Clathrin is a multimeric protein involved in vesicle coat assembly. Recently clathrin distribution was reported to change during the cell cycle and was found to associate with the mitotic spindle. Here we test whether the recruitment of clathrin to the spindle is indicative of a critical functional contribution to mitosis.

METHODOLOGY/PRINCIPAL FINDINGS: Previously a chicken pre-B lymphoma cell line (DKO-R) was developed in which the endogenous clathrin heavy chain alleles were replaced with the human clathrin heavy chain under the control of a tetracycline-regulatable promoter. Receptor-mediated and fluid-phase endocytosis were significantly inhibited in this line following clathrin knockout, and we used this to explore the significance of clathrin heavy chain expression for cell cycle progression. We confirmed using confocal microscopy that clathrin colocalised with tubulin at mitotic spindles. Using a propidium iodide flow cytometric assay we found no statistical difference in the cell cycle distribution of the knockout cells versus the wild-type. Additionally, we showed that the ploidy and the recovery kinetics following cell cycle arrest with nocodazole were unchanged by repressing clathrin heavy chain expression.

CONCLUSIONS/SIGNIFICANCE: We conclude that the association of clathrin with the mitotic spindle and the contribution of clathrin to endocytosis are evolutionarily conserved. However we find that the contribution of clathrin to mitosis is less robust and dependent on cellular context. In other cell-lines silencing RNA has been used by others to knockdown clathrin expression resulting in an increase in the mitotic index of the cells. We show an effect on the G2/M phase population of clathrin knockdown in HEK293 cells but show that repressing clathrin expression in the DKO-R cell-line has no effect on the size of this population. Consequently this work highlights the need for a more detailed molecular understanding of the recruitment and function of clathrin at the spindle, since the localisation but not the impact of clathrin on mitosis appears to be robust in plants, mammalian and chicken B-cells.